Abstract Mitochondria are ubiquitous organelles in eukaryotic cells that play important roles in energy production, metabolism, signal transduction and cell death. These functions require precise control of mitochondrial division, and altered mitochondrial division has been linked to many neurological diseases. A key protein involved in mitochondrial division is dynamin-related protein 1 (Drp1), a mechano-chemical GTPase that constitutes the mitochondrial division machinery. Drp1 is a soluble protein recruited to mitochondria from the cytosol by Drp1 receptor proteins located on mitochondria. After being recruited, Drp1 polymerizes into higher- order oligomers. Drp1 oligomers then drive the constriction of mitochondria. In the textbook model, mitochondrial division is regulated when Drp1 is recruited and oligomerized onto mitochondria. In contrast to this current view, our recent work suggested a major mechanism by which the timing of the constriction is regulated after Drp1 is oligomerized on mitochondria. This new mechanism involves novel interactions of Drp1 with the signaling phospholipid phosphatidic acid (PA) along with saturated phospholipids in the mitochondrial outer membrane. We suggest that these lipid interactions inhibit the GTPase activity of Drp1 oligomers and thereby control the initiation of the membrane constriction. In addition, we found that a mitochondrial, PA-producing phospholipase D, MitoPLD, directly binds Drp1 and inhibits mitochondrial division. This result suggests that PA is locally created in the vicinity of the division machinery. This local PA production may ensure robust spatial regulation of Drp1. These findings led to the hypothesis that PA inhibits Drp1 and the dissociation of Drp1 from MitoPLD activates mitochondrial division. In this proposed study, we will critically test this hypothesis and further develop and adjust it in an informed way. In Aim 1, we will determine how PA changes Drp1 activity using innovative biochemical, biophysical and cellular assays. We will also analyze how the mitochondrial lipid composition changes during division using lipidomics. In Aim 2, we will decipher how Drp1-MitoPLD interactions modulate the activity of both Drp1 and MitoPLD in mitochondrial division and how Drp1-MitoPLD interactions are regulated. We will also determine how MitoPLD controls the lipid composition in mitochondria. We expect that the outcomes of this proposed study will significantly advance the important biology of phospholipids and organelle dynamics and produce critical insights into human health and diseases.